CN117310012A

CN117310012A - Minocycline hydrochloride and high performance liquid chromatography detection method for impurities thereof

Info

Publication number: CN117310012A
Application number: CN202311054210.4A
Authority: CN
Inventors: 吴频平; 郑金琪; 俞育庆; 陶巧凤; 曹瑞伟; 朱坤丹; 陈彩萍; 周巧丽; 曾红霞; 翁晶
Original assignee: Zhejiang Changhai Pharmaceuticals Co ltd; ZHEJIANG INSTITUTE FOR FOOD AND DRUG CONTROL; Zhejiang Medicine Co Ltd
Current assignee: Zhejiang Changhai Pharmaceuticals Co ltd; ZHEJIANG INSTITUTE FOR FOOD AND DRUG CONTROL; Zhejiang Medicine Co Ltd
Priority date: 2023-08-21
Filing date: 2023-08-21
Publication date: 2023-12-29

Abstract

The invention relates to a high performance liquid chromatography detection method for minocycline hydrochloride and impurities thereof. A method for determining related substances in minocycline hydrochloride, comprising the following operations: preparing sample solution, reference solution, system applicability solution and sensitivity solution according to specification of high performance liquid chromatography, and using octadecylsilane chemically bonded silica gel as filler; taking ammonium acetate solution-methanol mixed solution containing disodium ethylenediamine tetraacetate as a mobile phase A, acetonitrile-methanol as a mobile phase B, and performing linear gradient elution, wherein the separation degree of minocycline peak and RS12 peak is not less than 1.5; the impurity peak corresponding to the relative retention time exists in the chromatogram of the sample solution, and the percentage of the impurity is calculated according to the external standard method of the main component reference substance added with the correction factors. The invention also provides minocycline hydrochloride. The method has the excellent technical effects as described in the specification.

Description

Minocycline hydrochloride and high performance liquid chromatography detection method for impurities thereof

Technical Field

The invention belongs to the technical field of medicines, relates to a quality analysis method of medicines, in particular to minocycline hydrochloride and impurities thereof, and more particularly relates to a method for detecting related substances in minocycline hydrochloride by using high performance liquid chromatography.

Background

Minocycline (minocycline), the hydrochloride salt of which is commonly used clinically. Minocycline hydrochloride has a molecular formula of C23H27N3 o7.hcl, a molecular weight of 493.94, and a chemical name: [4S- (4 alpha, 5 alpha, 12 alpha) ] -4, 7-bis (dimethylamino) -1, 4a, 5a,6,11,12 a-octahydro-3, 10,12 a-tetrahydroxy-1, 11-dioxo-2-tetracarboxamide hydrochloride, having the chemical structural formula:

minocycline hydrochloride is yellow crystalline powder, odorless and moisture-absorbing; dissolved in methanol, slightly dissolved in water, slightly dissolved in ethanol, and hardly dissolved in diethyl ether.

Minocycline belongs to a second-generation tetracycline antibiotic, is a long-acting and efficient semisynthetic tetracycline antibiotic, has an antibacterial spectrum similar to that of tetracyclines, has the strongest antibacterial effect in tetracyclines, has 2-4 times stronger activity against gram-positive bacteria than tetracyclines, has strong activity against both gram-positive bacteria and gram-negative bacteria, has cross drug resistance between tetracycline antibiotics, but has strong antibacterial activity against clinically separated tetracycline-resistant golden grapes, epizoons and streptococcus, and is clinically favored because of being effective against drug-resistant bacteria. Adverse reactions are substantially the same as other tetracyclines and can cause reversible vestibular reactions, including nausea, vomiting, and movement disorders.

Taking minocycline hydrochloride as an example, minocycline hydrochloride is taken up in the chinese pharmacopoeia 2020 edition, the united states pharmacopeia USP (date of validation is 5 months 1 day in 2020), the european pharmacopeia (EP 11.0) and the japanese pharmacopeia (JP 18).

The mobile phase system of ammonium acetate-dimethylformamide-tetrahydrofuran-ethylenediamine tetraacetic acid and the C8 chromatographic system are adopted in the 2020 edition of Chinese pharmacopoeia to control related substances, wherein the mainly controlled impurities comprise differential minocycline (< 1.2%), other single impurities (< 1.2%) and other total impurities (< 2.0%). In the current pharmacopoeias of USP, EP and JP, the mobile phase system of the method for detecting substances related to minocycline hydrochloride bulk drug is as follows: the oxalate-dimethylformamide-tetrahydrofuran-ethylenediamine tetraacetic acid disodium system controls epimers, other single impurities and total amount of impurities.

However, on the one hand, in face of increasingly stringent control requirements on impurities of substances, the control of specific impurities in most enterprises is not limited to epimers, and it is often necessary to separate and detect several or even more than ten specific impurities in order to better control the quality of the product. However, using the analytical methods in the current standard, the peak of the differential minocycline, other impurities, and minocycline peak, as well as some of the impurity peaks, cannot be separated effectively. Therefore, development of an analytical detection method with better specificity and selectivity is required, and effective separation of complex specific impurities and unknown impurities is achieved.

With the development of society and the increasing importance of human health and safety, the use of green analytical chemistry concepts to reduce the use of some toxic and harmful reagents has become a necessary trend in the experimental process. At present, in the detection process of related substances of tetracycline antibiotics, the mainstream detection method still uses toxic and harmful reagents such as tetrahydrofuran, dimethylformamide and the like when preparing a mobile phase, so that the body of an experimenter is chronically damaged, and the discharge of waste liquid also aggravates environmental pollution. In order to ensure the health of the analyst and reduce the environmental pollution problem, the use of the reagent should be avoided as much as possible, or when the organic reagent is unavoidable, the reagent is replaced by low toxicity.

Currently, there are related research teams that have made progress in conducting research on low toxicity solvent replacement. For example, gao Yanxia group [ Gao Yanxia, jiang Jianguo, du Zenghui ] HPLC method for determining minocycline hydrochloride for injection and related substances [ J ]. J.Chinese J.antibiotics, 2005, 30 (12): 744-746 reports that when using a Kromasil C8 (4.6 mm. Times.250 mm,5 μm) column, using 0.2mol/L ammonium acetate: N, N-dimethylformamide: tetrahydrofuran (600:398:2, containing 0.01mol/L disodium ethylenediamine tetraacetate) as a mobile phase, the detection wavelength was 280nm, and when detecting minocycline hydrochloride-related substances, 5 kinds of impurities were able to be detected: effective separation and control of desmethylaureomycin, 6-Desmethyltetracycline (DMTC), 6-desmethyl-6-deoxytetracycline (DMDOTC), 11 a-chloro-6-desmethyl-6-deoxytetracycline (11 a-C-l DMDOTC), 7-amino-6-desmethyl-6-deoxytetracycline (ADMDOTC). The method adopts acetate with better water solubility, reduces salt concentration, avoids using oxalate (which is crystallized and separated out in a low-temperature environment, and is easy to damage an instrument and a chromatographic column), and has better applicability, however, the literature method only controls 5 specific impurities, and cannot meet the control requirement of related substances on a plurality of complex specific impurities.

Wang Long [ Wang Long, wang Gongmei ] high performance liquid chromatography to determine minocycline hydrochloride related substances [ J ]. Strait pharmacy, 2006, 18 (12006): 85-86], using a Kromasil C8 column (250 mm. Times.4.6 mm,5 μm), adjusting pH6.55 with concentrated ammonia or acetic acid using ammonium acetate buffer-acetonitrile-methanol (830:170:5) as mobile phase, detecting the wavelength of 280nm, for 8 impurities that may be present in minocycline hydrochloride: (1) 7-didemethylminocycline, (2) demethylcycline, (3) dimetocycline, (4)7-monodemethylminocycline, (5) pyrazolominocycline, (6) demethylcycline, (7)4-differential minocycline, and (8) hydroxymethyl minocycline are separated and measured, so that the detection requirements of the 8 impurity controls are met, and methanol and acetonitrile are used for replacing tetrahydrofuran as organic phases, so that the damage of tetrahydrofuran to human bodies is reduced.

Quality evaluation of minocycline hydrochloride capsules [ J ] chinese journal of antibiotics, 2020, 45 (3): 224-233], using a chromatographic column Dikma Platisil ODS (250 mm. Times.4.6 mm,5 μm); the mobile phase A is 0.05mol/L ammonium acetate solution (the pH value is adjusted to 8.0 by ammonia water), the mobile phase B is methanol, the mobile phase C is acetonitrile, and the gradient elution procedure is carried out at the flow rate of 1.0mL/min; an ultraviolet detector for detecting a wavelength of 280nm; column temperature is 30 ℃; and the sample injection amount is 10 mu L, and related substances in minocycline are detected. Compared with the current pharmacopoeia method, the method has the advantages of lower column pressure, better separation effect among impurities, more detection of 3 impurities, more stable system, and effective separation of 10 unknown impurities except for the detection of the differential minocycline impurities.

However, with the continuous improvement of the separation technology, more specific impurities are separated and purified, the requirements of enterprises on impurity control are increasingly strict, the types of specific impurities are increased to more than ten kinds, more than twenty kinds, the limit is tightened, and the like, the impurities are difficult to completely separate by means of the existing detection means, and further, a new analysis method is required to be developed by continuing improvement and optimization, so that the separation and detection capability of the impurities in the detection of related substances are improved. At present, most chromatographic columns used for detecting minocycline hydrochloride bulk drugs in China are octadecylsilane chemically bonded silica (C18) or octyl silane chemically bonded silica (C8) chromatographic columns, and the phenomena of peak broadening, tailing and the like are easy to occur.

However, there is still a need in the art for new methods for detecting related substances in minocycline hydrochloride, for example, it is expected that the methods can increase the column efficiency, improve the separation effect between impurities, provide chromatographic columns with higher throughput and higher column efficiency, and provide a new and beneficial choice for the development of antibiotic related substance methods.

Disclosure of Invention

The present invention aims to provide a method for determining substances of interest in minocycline hydrochloride, which is expected to exhibit one or more beneficial effects, such as an accurate and reliable determination method, low cost of labor protection, etc.

To this end, the first aspect of the invention provides a method for determining substances of interest in minocycline hydrochloride, comprising the following operations:

(1) Performing according to specification of high performance liquid chromatography, and preparing each test solution;

(2) Preparation of the test solution:

test solution: taking a proper amount of test sample, adding water for dissolution and diluting to prepare a solution containing about 1mg of minocycline in 1 ml;

control solution: taking a proper amount of minocycline hydrochloride reference substance, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution with about 5 mug in each 1 ml;

system applicability solution: taking a proper amount of minocycline hydrochloride system applicability reference substance, adding water for dissolution and quantitatively diluting to prepare a solution containing about 1mg in each 1 ml;

sensitivity solution: precisely measuring a proper amount of reference substance solution, and quantitatively diluting with water to prepare a solution with about 0.5 mug in each 1 ml;

(3) Chromatographic conditions:

chromatographic column with octadecylsilane chemically bonded silica as filler; a mixed solution of 0.25mol/L ammonium acetate solution-methanol (90:10) containing 0.002mol/L disodium ethylenediamine tetraacetate (pH value is adjusted to 6.75+/-0.05 by ammonia water or glacial acetic acid) is used as a mobile phase A, acetonitrile-methanol (90:10) is used as a mobile phase B, and linear gradient elution is carried out according to the following table, wherein the flow rate is 1.0ml per minute; the column temperature is 35 ℃; the detection wavelength is 280nm; the temperature of the sample tray is controlled to be 2-8 ℃, and the sample injection volume is 10 μl;

time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0	95	5
12	92	8
			22	92	8
42	85	15
			50	75	25
60	75	25
			60.1	95	5
70	95	5

System applicability requirements: in the system applicability solution chromatogram, the separation degree of minocycline peak and RS12 peak is not less than 1.5; in the sensitivity solution chromatogram, the signal to noise ratio of a main peak is not less than 10; continuously sampling the reference substance solution for 6 times, wherein the relative standard deviation of minocycline peak area is not more than 5.0%;

(4) The percentage of impurities was determined and calculated: precisely measuring the sample solution and the reference solution, respectively injecting into a liquid chromatograph, recording chromatogram, calculating the percentage of the impurity according to the external standard method of the main component reference with correction factors, wherein the impurity peak corresponds to the Relative Retention Time (RRT) as shown in the table in the chromatogram of the sample solution,

the method according to the first aspect of the present invention, wherein the specification of the high performance liquid chromatography is a specification of high performance liquid chromatography carried in the chinese pharmacopoeia or the united states pharmacopoeia or the european pharmacopoeia or the japanese pharmacopoeia of the calendar version, for example, a specification of the fourth edition general rule 0512 high performance liquid chromatography of the chinese pharmacopoeia 2020.

The method according to the first aspect of the present invention, wherein the minocycline hydrochloride system applicability control comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11, and minocycline.

The method according to the first aspect of the present invention, wherein the minocycline hydrochloride system applicability control comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11, minocycline, and further comprises impurities RS17, RS28, RS30, RS33, and RS40, and has the structural formula and/or chemical name:

impurity RS17:

(4S, 4aS,5aR,12 aS) -9-amino-4- (dimethylamino) -3,10,12 a-tetrahydroxy-1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide (9-amino mountain Cyclo-ine),

impurity RS28 or 9-aminominocycline:

impurity RS30 or monomethyl ethyl minocycline:

impurity RS33:

(4S, 4aS,5aR,12 aS) -7-amino-4- (dimethylamino) -3,10,12 a-tetrahydroxy-1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide (7-amino mountain Cyclo-ine),

impurity RS40 or norchlortetracycline:

the method according to the first aspect of the invention, wherein the octadecylsilane chemically bonded silica is packed in a Agilent InfinityLab Poroshell HPH-C18 column having a size of 4.6mm.times.150mm, 2.7 μm, or other column of comparable performance.

The method according to the first aspect of the present invention, wherein minocycline hydrochloride is considered acceptable when it is detected that it contains impurities that meet the following limit specifications: not more than 1.2% of RS03, not more than 0.8% of RS32, not more than 0.6% of RS02, not more than 0.6% of RS11, not more than 0.5% of RS12, not more than 0.5% of RS15, not more than 0.3% of RS16, not more than 0.10% of other single impurities, not more than 2.0% of the total amount of impurities (except RS 03), and ignoring peaks (0.05%) smaller than the main peak area of the sensitivity solution.

The method according to the first aspect of the invention, wherein the theoretical plate number of minocycline peaks is greater than 1 ten thousand, e.g. greater than 2 ten thousand, e.g. greater than 5 ten thousand.

According to the method of the first aspect of the invention, the minocycline hydrochloride system applicability reference substance comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and the separation degree between each two adjacent peaks is greater than 1.5.

According to the method of the first aspect of the invention, the minocycline hydrochloride system applicability control comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and also comprises impurities RS17, RS28, RS30, RS33 and RS40, wherein the separation degree between adjacent peaks is greater than 1.5.

The method according to the first aspect of the present invention, wherein minocycline hydrochloride is considered acceptable when it is detected that it contains impurities that meet the following limit specifications: the impurity RS17 is not more than 0.2%, for example not more than 0.10%, the impurity RS28 is not more than 0.2%, for example not more than 0.10%, the impurity RS30 is not more than 0.2%, for example not more than 0.10%, the impurity RS33 is not more than 0.2%, for example not more than 0.10%, and the impurity RS40 is not more than 0.2%, for example not more than 0.10%.

The method according to the first aspect of the invention, wherein the degree of separation between minocycline and adjacent impurities in the chromatogram of the test solution is greater than 2.0

Further, the second aspect of the present invention provides minocycline hydrochloride, which is used for determining related substances therein by the method according to any one of the embodiments of the first aspect of the present invention, wherein the minocycline hydrochloride is detected as impurities defined by the following limits: no more than 1.2% RS03, no more than 0.8% RS32, no more than 0.6% RS02, no more than 0.6% RS11, no more than 0.5% RS12, no more than 0.5% RS15, no more than 0.3% RS16, no more than 0.10% other individual impurities, no more than 2.0% total impurities (excluding RS 03), peaks less than the major peak area of the sensitivity solution (0.05%) are negligible.

Minocycline hydrochloride according to the second aspect of the present invention, in which impurities having the following limits are detected, is determined using the method according to any one of the embodiments of the first aspect of the present invention: no more than 0.2%, such as no more than 0.10%, of impurity RS17, no more than 0.2%, such as no more than 0.10%, of impurity RS28, no more than 0.2%, such as no more than 0.1%, of impurity RS30, no more than 0.2%, such as no more than 0.10%, of impurity RS33, no more than 0.2%, such as no more than 0.10%, of impurity RS40.

The method of the invention achieves some good effects. For example, the method of the invention has a large quantity of separated impurities, can effectively separate and detect 12 specific impurities, and has a degree of separation between the impurities and the main peak of more than 1.5; the problems of poor separation degree, low recovery rate and poor solution stability of impurities are solved; the invention selects a specific liquid chromatographic column, improves the separation degree between unknown impurities, solves the problem that the combination integral is difficult due to poor separation of individual unknown single impurities, and the limit of single impurities is difficult to tighten, and can be tightened to 0.10% from 0.15%; the mobile phase of the method does not contain substances with larger toxicity such as N, N-dimethylformamide, tetrahydrofuran and the like, is environment-friendly, reduces the damage to human bodies, improves the stability of instruments and chromatographic systems, and has high safety, simple and convenient operation and low cost.

Drawings

Fig. 1: typical HPLC profile of a system-adapted solution containing 7 typical impurities and minocycline.

Fig. 2: typical chromatograms of impurity mixtures comprising 12 impurities and minocycline and their integral data.

Fig. 3: a typical chromatogram of an impurity mixture comprising 12 impurities and minocycline is a partial detail view over a period of 35-55 min.

Fig. 4: typical chromatograms of a mixed liquor containing 12 impurities and minocycline at low concentration.

Fig. 5: typical test solution HPLC profiles of three samples measured using the method of example 1 of the present invention are FIG. 5A, FIG. 5B, and FIG. 5C, respectively.

Detailed Description

The present invention is further illustrated by the following specific examples, in which various reagents and apparatus are readily available from the market unless otherwise specified.

Example 1: method for determining related substances in minocycline hydrochloride

(1) According to the specification of the high performance liquid chromatography of the four general rules 0512 of the 2020 edition of Chinese pharmacopoeia, each test solution is prepared newly;

(2) Preparation of the test solution:

system applicability solution: taking a proper amount of minocycline hydrochloride system applicability reference substance (which accords with EP11.0 and contains RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline), adding water for dissolving and quantitatively diluting to prepare a solution containing about 1mg in each 1 ml;

(3) Chromatographic conditions:

chromatography columns packed with octadecylsilane-bonded silica gel (Agilent InfinityLab Poroshell HPH-C18 column, 4.6 mm. Times.150 mm,2.7 μm in this example; other columns believed to be equivalent in performance may also be used); a0.25 mol/L ammonium acetate solution-methanol (90:10) mixture (pH value is adjusted to 6.75+ -0.05 by ammonia water or glacial acetic acid) containing 0.002mol/L disodium ethylenediamine tetraacetate is used as mobile phase A, acetonitrile-methanol (90:10) is used as mobile phase B, and linear gradient elution is carried out according to the following table (Table 1) at a flow rate of 1.0ml per minute; the column temperature is 35 ℃; the detection wavelength is 280nm; the temperature of the sample tray is controlled to be 2-8 ℃, and the sample injection volume is 10 μl;

table 1:

(4) The percentage of impurities was determined and calculated: precisely measuring the sample solution and the reference solution, respectively injecting into a liquid chromatograph, recording chromatogram, calculating the percentage of the impurity according to the external standard method of the main component reference with correction factors, wherein the impurity peak corresponds to the Relative Retention Time (RRT) as shown in the table (table 2) in the chromatogram of the sample solution,

table 2:

according to the detection of some typical minocycline hydrochloride samples by the inventor, the general limits of certain impurities can be generally determined, for example, according to the test and calculation results of this embodiment, if there are impurity peaks in the chromatogram of the sample solution, the sample solution is calculated according to the external standard method of the main component control added with correction factors, and the sample solution contains:

RS03 may generally specify less than 1.2%,

RS32 may generally specify less than 0.8%,

RS02 may generally specify less than 0.6%,

RS11 may generally specify less than 0.6%,

RS12 may generally specify less than 0.5%,

RS15 may generally specify less than 0.5%,

RS16 may generally specify less than 0.3%,

other individual impurities may be generally prescribed to be less than 0.10%, the total amount of impurities (other than RS 03) may be generally prescribed to be less than 2.0%, and peaks (0.05%) smaller than the main peak area of the sensitivity solution may be generally prescribed to be negligible. The general limit results obtained above, namely, the provision of: not more than 1.2% of RS03, not more than 0.8% of RS32, not more than 0.6% of RS02, not more than 0.6% of RS11, not more than 0.5% of RS12, not more than 0.5% of RS15, not more than 0.3% of RS16, not more than 0.10% of other single impurities, not more than 2.0% of the total amount of impurities (except RS 03), and ignoring peaks (0.05%) smaller than the main peak area of the sensitivity solution.

In this example, the chemical names and chemical structural formulas of the impurities listed in table 2 are as follows:

ep.a: (4R, 4aS,5aR,12 aS) -4, 7-bis (dimethylamino) -3,10,12 a-tetrahydroxy-1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide (4-epi minocycline),

ep.b: (4S, 4aS,5aR,12 aS) -4- (dimethylamino) -3,10,12 a-tetrahydroxy-1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide (mountain cyclic lactone),

ep.c: (4S, 4aS,5aR,12 aS) -4- (dimethylamino) -3,10,12 a-tetrahydroxy-7- (methylamino) -1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide (7-monodemethylminocycline),

ep.e: (4S, 4aS,5aR,12 aS) -4, 7-bis (dimethylamino) -3,10,12 a-trihydroxy-12-imino-1, 11-dioxo-1, 4a, 5a,6,11 a,12 a-decahydrotetracene-2-carboxamide,

ep.f: (4S, 4aS,5aR,12 aS) -4, 7-bis (dimethylamino) -3,10,12 a-tetrahydroxy-N- (hydroxymethyl) -1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide,

ep.g: (4S, 4aS,5aR,12 aS) -4,7, 9-tris (dimethylamino) -3,10,12 a-tetrahydroxy-1, 11-dioxo-1, 4a, 5a,6,11,12 a-octahydrotetracene-2-carboxamide,

ep.h: (4S, 4aS,12 aS) -4, 7-bis (dimethylamino) -3,10,11,12 a-tetrahydroxy-1, 12-dioxo-1, 4a,5,12 a-hexahydrotetracene-2-carboxamide,

in this example, a typical HPLC diagram of the system-suitable solution is shown in fig. 1, wherein the retention time of minocycline as the main component is about 45.5min, the separation degree of each impurity from the minocycline peak as the main component is greater than 1.5, for example, the separation degree between the minocycline peak as the main component and the closest impurity RS12 peak is 2.3, and the separation degree between the remaining impurities is also greater than 1.5. This shows that the high performance liquid chromatography of this example can be effectively used for determining typical impurities in minocycline hydrochloride, and the determination conditions have the advantage of being green and environment-friendly.

Example 2: determination of certain specific impurities of minocycline hydrochloride

It should be noted in particular that in the method of example 1 above, the usual impurities in minocycline hydrochloride are effectively separated from 7 impurities as shown in table 2 above using green chromatographic conditions and are effectively quantified. However, it is known that, depending on the preparation method of the crude drug and its physical and chemical properties, minocycline hydrochloride also contains some other relatively common impurities, such as the following impurities RS17, RS28, RS30, RS33, and RS40:

impurity RS17 or impurity ep.i:

impurity RS28 or 9-aminominocycline:

impurity RS30 or monomethyl ethyl minocycline:

impurity RS33 or impurity ep.d:

impurity RS40 or norchlortetracycline:

the present inventors have found that, using the method of example 1, in addition to the simultaneous separation and measurement of 7 impurities therein, the above 5 impurities can be simultaneously measured, so that 13 substances including minocycline as a main component can be separated and measured under the same chromatographic conditions, as follows.

Reference example 1 was made; taking proper amounts of impurity RS17, impurity RS28, impurity RS30, impurity RS33 and impurity RS40, adding water to dissolve and dilute to prepare a solution containing 0.2-1 mg of each impurity in each 1ml, taking the solution as a stock solution, and taking the stock solution and the system applicability solution described in the example 1 according to a ratio of 5:95 to obtain an impurity mixed solution, injecting the impurity mixed solution into a liquid chromatograph for measurement according to the method of the embodiment 1, and recording a chromatogram; in this experiment, a typical HPLC diagram of the impurity mixed solution is shown in fig. 2, and a partial detail of the impurity mixed solution during 35 min-55 min is shown in fig. 3, wherein the retention time of the minocycline serving as a main component is about 45.5min, the separation degree of each impurity from the minocycline peak serving as a main component is greater than 1.5, for example, the separation degree between the minocycline peak serving as a main component and the peak of the closest impurity RS12 reaches 2.3, and the separation degree between the other impurities is also greater than 1.5; the number of theoretical plates of each peak is 5000 or more, and especially the number of theoretical plates of the main component is 14 ten thousand or more. This shows that the high performance liquid chromatography of example 1 of the present invention can be effectively used to determine up to 12 typical impurities in minocycline hydrochloride.

Further, the process was carried out with reference to example 1; taking 7 impurities of the embodiment 1 and proper amounts of the impurities RS17, the impurity RS28, the impurity RS30, the impurity RS33, the impurity RS40 and minocycline hydrochloride reference substances, adding water for dissolving and diluting to prepare a solution containing 1-10 mug of each impurity and minocycline in 1ml, wherein the concentration of minocycline in the solution is low as an impurity mixed reference substance solution, so that each peak and separation condition can be conveniently identified; taking the impurity mixed reference substance solution, injecting the impurity mixed reference substance solution into a liquid chromatograph for measurement according to the method of the embodiment 1, and recording a chromatogram; in this experiment, a typical HPLC diagram of the impurity mixed reference solution is shown in fig. 4, wherein the retention time of the minocycline main component is about 45.4min, the separation degree of each impurity from the minocycline main component peak is greater than 1.5, for example, the separation degree between the minocycline main component peak and the closest impurity RS12 peak is up to 2.3, and the separation degree between the other impurities is also greater than 1.5. This shows that the high performance liquid chromatography of example 1 of the present invention can be effectively used to determine up to 12 typical impurities in minocycline hydrochloride.

Example 3: performance investigation of method for determining minocycline hydrochloride related substances

Examples 1 and 2 of the present invention show that the method can be effectively used for measuring a related substance in minocycline hydrochloride and exhibits one or more excellent effects. This example 3 was subjected to methodological performance investigation using conventional pharmaceutical analysis methods well known to those skilled in the art, with respect to the methods of examples 1 and 2, the main results being summarized below.

1. Verification result of specificity and system applicability

(1) The blank solution has no interference;

(2) The system applicability meets the requirements;

(3) In the labeled sample solution, the minimum separation degree between minocycline and adjacent impurities is 2.2;

(4) Under the condition of forced degradation, minocycline and impurity peaks can be well separated, the minocycline peak purity factors are larger than 999, and the mass balance is within the range of 95-105%. The results of the above 4 items all meet the general quality detection requirements in the field.

2. Verification of quantitative limits

The signal to noise ratio of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and minocycline is usually required to be not less than 10, and the peak area RSD is usually required to be not more than 10.0% by repeating sample injection 6 times by using the prepared quantitative limiting solution. As a result, the quantitative limit concentrations of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and minocycline are all in the range of 0.4-0.5 μg/ml, for example, the quantitative limit concentration of minocycline is measured to be 0.465 μg/ml, the signal to noise ratio is greater than 10, and the peak area RSD is less than 10.0%.

3. Verification result of detection limit

The prepared detection limit solution is used for repeated sampling for 3 times, and the signal to noise ratio of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and minocycline is prepared to be not less than 3. As a result, the detection limit concentrations of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and minocycline are all in the range of 0.21-0.26 μg/ml, for example, the quantitative limit concentration of minocycline is measured to be 0.233 μg/ml, and the signal to noise ratio is greater than 3.

4. Verification of linearity and range

As measured, RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 are in the range of 50% -150% (relative to the limiting concentration), minocycline is in the concentration range of 2.323 μg/ml to 6.970 μg/ml, the correlation coefficients are all greater than 0.9999, and the Y-axis intercept absolute value generally requires less than 10% of the 100% linear concentration level response (measured is less than 6.6%).

5. Verification of accuracy results

According to measurement, the recovery rates of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33 and RS40 in the range of 50-150% (relative to the limit concentration) are all between 90-110%, and the RSD values are all less than 5.0%; for example, the average recovery rate of each impurity is in the range of 92 to 103%.

6. Verification of precision

In the repeatability test, an analyzer A prepares 6 parts of test solution in parallel, and the test is carried out according to law: comparing 6 detection results, wherein the maximum single impurity range of RS02, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and other impurities is less than 0.05%; RSD of RS03 and total impurities (excluding RS 03) were 1.4% and 0.8%, respectively.

6. Verification of solution stability

The control solution and the test solution are placed for a period of time at the temperature of 2-8 ℃, and then the detection results at each time point are compared with the results of 0h, as a result, (1) the control solution is placed for 22h at the temperature of 2-8 ℃ and then is detected, and compared with 0h, the RD value of the main peak area is less than 2.9%; (2) The sample solution is detected after being placed for 54 hours at the temperature of 2-8 ℃, and compared with 0 hour, the absolute difference of the detection results of the sample solution is less than 0.25 percent (RS 02, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and other maximum single impurities) respectively is less than 0.05 percent; RD values of RS03 and total impurities (excluding RS 03) were 9.8% and 8.3%, respectively. It has been found that when the ammonium acetate used in the mobile phase of the process of example 1 is changed to the same concentration of ammonium oxalate, the RD values of RS03 and total impurities (excluding RS 03) are 21.2% and 16.8%, respectively, indicating that ammonium acetate is not suitable to be replaced arbitrarily.

7. Durability verification results

Respectively changing the flow rate, the column temperature and the pH value of the mobile phase: (1) the system applicability meets the requirements; (2) Compared with the detection result under the standard condition, the absolute difference of less than 0.25 percent of impurities (RS 02, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and other maximum single impurities) is less than 0.05 percent; RS03 and total impurities (excluding RS 03) RD were 5.9% and 4.2%, respectively.

All the verification results completely meet the requirements of a common analysis method.

Example 4: test method and selection of conditions

The methods for determining the relevant substances in minocycline hydrochloride provided in examples 1 and 2, summarized after a great inventive effort, have found that a unique choice of chromatographic column and mobile phase is essential for successful efficient separation of 12 impurities, some of which occur as well as not foreseen in the prior art, and are specifically tested as follows. Referring to the method of example 1, the only difference is the following make/model chromatographic column: sunniest C18 column (4.6X105 mm,5 μm), inertsustein C8 column (4.6X105 mm,5 μm), waters Symmetey C column (4.6X105 mm,5 μm), agilent InfinityLab Poroshell Aq-C18 column (4.6X105 mm,2.7 μm), agilent InfinityLab Poroshell 120CS-C18 column (4.6X105 mm,2.7 μm), agilent InfinityLab Poroshell 120SB-C18 column (4.6X105 mm,2.7 μm), and as a result, the effective separation of all 12 kinds of impurities could not be achieved; specifically: the Sunniest C18 column, the Inertsustein C8 column and the Waters Symmetey C column can not effectively separate the RS02, the RS40 and the RS16 (the separation degree between adjacent peaks is less than 0.8), the RS12 peak is hidden inside the minocycline peak and can not separate the two peaks, and the separation degree between the RS15 peak and the minocycline peak is in the range of 0.9-1.1, for example, when the Sunniest C18 column is used, the separation degree between the RS02 and the RS40 is only 0.56 and the separation degree between the RS15 peak and the minocycline peak is 1.03; all of the three Agilent InfinityLab Poroshell columns cannot separate RS02 from RS40 (the separation degree is in the range of 0.7-1.0), and all cannot separate the peak of RS12 from the peak of minocycline (the separation degree is in the range of 0.8-1.0), for example, when the Agilent InfinityLab Poroshell SB-C18 column is separated, the separation degree of RS02 from RS40 is 0.84, and the separation degree of the peak of RS12 from the peak of minocycline is 0.92; in general, when a chemical substance is separated and analyzed by reverse phase HPLC, the degree of separation between peaks is required to be 1.5 or more, however, the inventors found that even with the Agilent InfinityLab Poroshell column of the same brand as in example 1 of the present invention, the separation effect is significantly inferior to that of the chromatographic column of example 1. With reference to the method of example 1, the only difference is that the mobile phases of the following four pharmacopoeia methods are respectively changed and elution is carried out in a mode of a flow rate of 1.0 ml/min: CP mobile phase: 0.2M ammonium acetate-dimethylformamide-tetrahydrofuran (600:398:2, containing 0.01mol/L disodium ethylenediamine tetraacetate), USP mobile phase: dimethylformamide-tetrahydrofuran-0.2M ammonium oxalate-0.01M disodium edentate (120:80:600:180, ammonium hydroxide adjusted ph=7.2), JP mobile phase: ammonium oxalate monohydrate (7 g/250 ml), dimethylformamide, 0.1M disodium ethylenediamine tetraacetate (11:5:4) were mixed with tetrabutylammonium hydroxide to adjust pH6.5, EP mobile phase: dimethylformamide-tetrahydrofuran-solution A (volume ratio: 12:8:78, solution A is a mixture of 18 volumes of 3.75g/L sodium edetate solution and 60 volumes of 28.3g/L ammonium oxalate solution and pH 7.2 is adjusted with ammonia water); when the conditions of example 1 of the present invention were used but the mobile phase was changed to the four isocratic elutions described above, the results could not achieve effective separation of all 12 impurities; specifically: the four mobile phases can not separate RS33 from RS17 (two peaks overlap), the three peaks of RS02, RS40 and RS16 can not achieve acceptable separation (the separation degree between two adjacent peaks is smaller than 0.7), the three peaks of RS12, minocycline and RS15 can not achieve acceptable separation (the separation degree between two adjacent peaks is smaller than 0.8 or RS12 is hidden in minocycline peak), for example, when a CP mobile phase is used, the two peaks of RS33 and RS17 overlap, the separation degree between the two peaks of RS02 and RS40 is 0.46, the separation degree between the two peaks of RS12 is hidden in minocycline peak, and the separation degree between the two peaks of minocycline and RS15 is 0.68; these results indicate that even with the chromatographic column of the invention, 12 impurities and minocycline cannot be separated using the prior art mobile phase. Referring to the method for checking related substances under the two minocycline hydrochloride varieties in the 2020 edition of Chinese pharmacopoeia, when the chromatographic column of the embodiment 1 of the invention is used for measuring the test solution containing 12 impurities and minocycline in the embodiment 2 of the invention, only 7 impurity peaks which can reach the separation degree from the adjacent peaks of more than 1.5 are displayed; referring to the examination method of the related substances under the japanese pharmacopoeia JP XVIII edition minocycline hydrochloride variety item but instead of the chromatographic column of example 1 of the present invention, when the test solution of the present invention comprising 12 impurities and minocycline as shown in fig. 2 was measured, only 5 impurity peaks were shown which could achieve a degree of separation from the adjacent peaks of 1.5 or more; referring to the examination method of the related substances under the item of the European pharmacopoeia 11.0 edition minocycline hydrochloride variety, but by measuring the test solution comprising 12 impurities and minocycline according to FIG. 2 of the present invention by using the column of example 1 of the present invention, only 6 impurity peaks reaching a separation degree of 1.5 or more from the adjacent peaks were shown. In addition, there are some documents that, when measuring the related substances in minocycline hydrochloride, about 1% of triethylamine is added to the mobile phase, the inventors also tried to add a small amount of triethylamine to the mobile phase to examine the separation effect, and found that the result was not acceptable at all, and a specific test was performed in this way; referring to the methods of examples 1 and 2, except that the mobile phase A was a mixed solution of 0.002mol/L disodium ethylenediamine tetraacetate and 1% triethylamine in 0.25mol/L ammonium acetate solution-methanol (90:10) (pH was adjusted to 6.75.+ -. 0.05 with ammonia or glacial acetic acid), and the other was unchanged, when the mixed solution of impurities used in FIG. 2 was measured for the formation of minocycline containing 12 impurities and minocycline, both of R12 and R15 were hidden in the minocycline peak, the degree of separation between the impurities R02 and R40 was 0.36, the degree of separation between the impurities R40 and R16 was 0.53, and the three degree of separation of impurities were not at all satisfactory for general measurement; thus, the addition of triethylamine in the mobile phase does not contribute to an effective separation analysis of the main component from the 12 impurities.

Example 5: determination of some minocycline hydrochloride samples using the methods of the present invention

In this example, the method of example 1 of the present invention was used to measure the substances of interest in minocycline hydrochloride samples (three batches, batch number MN2102002B, MN2102003B, MN2102003B, respectively), and the results were obtained by measuring the substances of interest in the same item in chinese pharmacopoeia of 2020 edition and european pharmacopoeia EP11.0 edition, and comparing the results.

The results of the measurement using the method of example 1 of the present invention are shown in Table 3 below, and typical sample solution HPLC diagrams of three batches of samples are shown in FIGS. 5A, 5B, and 5C, respectively.

Table 3:

the three samples also detected 0.03-0.09% of impurities RS17, RS28, RS30, RS33, RS40, e.g., MN2102001B also detected 0.04% of RS17, 0.09% of RS28, 0.03% of RS17, 0.06% of RS17, 0.05% of RS17.

The results of the measurement using the method under the item of the European pharmacopoeia EP11.0 edition minocycline hydrochloride related substances are shown in Table 4 below.

Table 4:

none of the three samples failed to separate the impurities RS17, RS28, RS30, RS33, RS40 of the present invention, possibly combined with the peaks of the above table, which were significantly larger than RS03 and RS16 of Table 4 for impurities A and H, possibly with individual other impurities hidden in the two impurities.

The results of the measurement using the method under the relevant matter item of minocycline hydrochloride in the chinese pharmacopoeia of 2020 edition are shown in the following table 5.

Table 5:

as can be seen from the table, the other maximum single impurity is significantly larger than table 3, indicating that this maximum single impurity may be one peak formed by a plurality of impurities, and that the impurities RS15, RS16, RS28, RS30, RS32, RS33, RS40 cannot be identified.

The results according to the context of the present invention show that the method of the present invention, in particular the method of example 1, has good specificity, detection and quantification limits, linearity and range, accuracy, precision and solution stability. The specificity test shows that the blank solution does not interfere with the detection of the target impurity peak, and the system applicability test meets the requirements, so that the method has good specificity. Stability tests show that the peak area change of each target impurity is less than 10% when each solution is placed for 22 hours at the temperature of 2-8 ℃, and the stability of the sample solution is good. The durability test result shows that the method has certain selectivity to column temperature, mobile phase pH value, mobile phase proportion and the like, and the parameters are fixed in a certain range in the analysis method. The verification result shows that the analysis method can effectively detect up to 12 impurities including RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33 and RS40 in minocycline hydrochloride.

While various aspects of the present invention have been described above, it should be understood that these examples are illustrative only and the scope of the present invention is not limited to such examples.

Claims

1. A method for determining related substances in minocycline hydrochloride, comprising the following operations:

(2) Preparation of the test solution:

(3) Chromatographic conditions:

2. the method according to claim 1, wherein the specification of the high performance liquid chromatography is a specification of high performance liquid chromatography carried in the chinese pharmacopoeia or the united states pharmacopoeia or the european pharmacopoeia or the japanese pharmacopoeia of the calendar edition, for example the specification of the chinese pharmacopoeia 2020 edition four-part rule 0512 high performance liquid chromatography.

3. The method according to claim 1, wherein minocycline hydrochloride system applicability controls comprise impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11, and minocycline.

4. The method according to claim 1, wherein the minocycline hydrochloride system applicability control comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and further comprises impurities RS17, RS28, RS30, RS33 and RS40, wherein the structural formula and/or chemical name of the minocycline hydrochloride system applicability control are respectively as follows:

impurity RS17:

impurity RS28 or 9-aminominocycline:

impurity RS30 or monomethyl ethyl minocycline:

impurity RS33:

impurity RS40 or norchlortetracycline:

5. the method according to claim 1, wherein the octadecylsilane chemically bonded silica is a Agilent InfinityLab Poroshell HPH-C18 column having a size of 4.6mm by 150mm,2.7 μm, or other column of comparable performance.

6. The method according to claim 1, wherein minocycline hydrochloride is considered acceptable when it is detected that it contains impurities that meet the following limits: not more than 1.2% of RS03, not more than 0.8% of RS32, not more than 0.6% of RS02, not more than 0.6% of RS11, not more than 0.5% of RS12, not more than 0.5% of RS15, not more than 0.3% of RS16, not more than 0.10% of other single impurities, not more than 2.0% of the total amount of impurities (except RS 03), and ignoring peaks (0.05%) smaller than the main peak area of the sensitivity solution.

7. The method according to claim 1, wherein the theoretical plate number of minocycline peaks is greater than 1 ten thousand, such as greater than 2 ten thousand, such as greater than 5 ten thousand.

8. The method according to claim 1, wherein:

the minocycline hydrochloride system applicability reference substance comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and the separation degree between every two adjacent peaks is more than 1.5;

the minocycline hydrochloride system applicability reference substance comprises impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11, minocycline, impurities RS17, impurities RS28, impurities RS30, impurities RS33 and impurities RS40, and the separation degree between every two adjacent peaks is more than 1.5;

minocycline hydrochloride is considered to be acceptable when it is detected that it contains impurities that meet the following limit specifications: not more than 0.2% for impurity RS17, e.g., not more than 0.1%, not more than 0.2% for impurity RS28, e.g., not more than 0.1%, not more than 0.2% for impurity RS30, e.g., not more than 0.1%, not more than 0.2% for impurity RS33, e.g., not more than 0.1%, not more than 0.2% for impurity RS40, e.g., not more than 0.1%; and/or the number of the groups of groups,

in the chromatogram of the sample solution, the separation degree between minocycline and adjacent impurities is more than 2.0.

9. Minocycline hydrochloride, in which the impurities defined by the following limits are detected, determined using the method according to any one of claims 1 to 8, for the substances of interest: no more than 1.2% RS03, no more than 0.8% RS32, no more than 0.6% RS02, no more than 0.6% RS11, no more than 0.5% RS12, no more than 0.5% RS15, no more than 0.3% RS16, no more than 0.10% other individual impurities, no more than 2.0% total impurities (excluding RS 03), peaks less than the major peak area of the sensitivity solution (0.05%) are negligible.

10. Minocycline hydrochloride according to claim 9, in which the relevant substances are determined using the method according to any one of claims 1 to 8, the minocycline hydrochloride being detected as impurities having the following limits: no more than 0.2%, such as no more than 0.10%, of impurity RS17, no more than 0.2%, such as no more than 0.10%, of impurity RS28, no more than 0.2%, such as no more than 0.10%, of impurity RS30, no more than 0.2%, such as no more than 0.10%, of impurity RS33, no more than 0.2%, such as no more than 0.10%, of impurity RS40.